Uses of aldose-1-epimerase (mutarotase) for diagnosis and therapy of inflammatory diseases and sepsis

ABSTRACT

Uses of aldose 1-epimerase (SEQ ID NO:3) from body fluids or body tissues in human and veterinary medicine as a marker peptide for diagnosis, for prognosis of the course and for monitoring of the course of inflammations and infections and/or as a target for therapeutically influencing the course of inflammations and/or infections.

The present invention relates to novel uses of the enzyme aldose1-epimerase (also known as mutarotase; frequently abbreviated below to“A1E” or simply “epimerase”) for the medical diagnosis and therapy ofinflammatory diseases and sepsis. It is based on the detection for thefirst time of greatly increased concentrations of aldose 1-epimerase inliver tissue of primates in which a sepsis or systemic inflammation hadbeen produced experimentally by toxin administration.

The present invention has its origin in intensive research work by theApplicant in relation to further improvements of the diagnosis andtherapy of inflammations and infections, in particular of inflammationsof infectious aetiology and sepsis.

Inflammations are defined very generally as certain physiologicallyreactions of an organism to different types of external effects, suchas, for example, injuries, burns, allergens, infections bymicroorganisms, such as bacteria and fungi and viruses, to foreigntissues which trigger rejection reactions, or to certain inflammatoryendogenous conditions of the body, for example in autoimmune diseasesand cancer. Inflammations may occur as harmless, localized reactions ofthe body but are also typical features of numerous serious chronic andacute diseases of individual tissues, organs, organ parts and tissueparts.

Local inflammations are generally part of the healthy immune reaction ofthe body to harmful effects and hence part of the life-preservingdefence mechanism of the body. If, however, inflammations are part of amisdirected reaction of the body to certain endogenous processes, suchas, for example, in autoimmune diseases, and/or are of a chronic nature,or if they achieve a systematic extent, as in the case of systemicinflammatory response syndrome (SIRS) or in the case of a severe sepsiscaused by infection, the physiological processes typical of inflammatoryreactions go out of control and become the actual, frequentlylife-threatening pathological process.

It is now known that the origin and the course of inflammatory processesare controlled by a considerable number of substances which arepredominantly of a protein or peptide nature or are accompanied by theoccurrence of certain biomolecules for a more or less limited time. Theendogenous substances involved in inflammatory reactions include inparticular those which may be counted among the cytokines, mediators,vasoactive substances, acute phase proteins and/or hormonal regulators.The inflammatory reaction is a complex physiological reaction in whichboth the endogenous substances activating the inflammatory process (e.g.TNF-α) and deactivating substances (e.g. interleukin-10) are involved.

In systemic inflammations, as in the case of a sepsis or of septicshock, the inflammation-specific reaction cascades spread in anuncontrolled manner over the whole body and become life-threatening inthe context of an excessive immune response. Regarding the currentknowledge about the occurrence and possible role of individual groups ofendogenous inflammation-specific substances, reference is made, forexample, to A. Beishuizen et al., “Endogenous Mediators in Sepsis andSeptic Shock”, Advances in Clinical Chemistry, Vol. 33, 1999, 55-131;and C. Gabay et al., “Acute Phase Proteins and Other Systemic Responsesto Inflammation”, The New England Journal of Medicine, Vol. 340, No. 6,1999, 448-454. Since the understanding of sepsis and related systemicinflammatory diseases, and hence also the recognized definitions, havechanged in recent years, reference is also made to K. Reinhart et al.,“Sepsis and septischer Schock” [Sepsis and septic shock], in:Intensivmedizin, Georg Thieme Verlag, Stuttgart, New York, 2001,756-760, where a modern definition of sepsis is given. In the context ofthe present Application, the terms sepsis and inflammatory diseases usedare based on the definitions given in the stated three references.

Whereas at least in Europe the systemic bacterial infection detectableby a positive blood culture long characterized the term sepsis, sepsisis now primarily understood as being systemic inflammation which iscaused by infection but, as a pathological process, has greatsimilarities to systemic inflammations which are triggered by othercauses. Said transformation in the understanding of sepsis has resultedin changes in diagnostic approaches. Thus, the direct detection ofbacterial pathogens was replaced or supplemented by complex monitoringof physiological parameters and, more recently, in particular by thedetection of certain endogenous substances involved in the sepsisprocess or in the inflammatory process, i.e. specific “biomarkers”.

Of the large number of mediators and acute phase proteins which areknown to be involved in an inflammatory process, the ones which aresuitable for diagnostic purposes are in particular those whoseoccurrence is very specific for inflammatory diseases or certain phasesof inflammatory diseases and whose concentrations change in a dramaticand diagnostically significant manner and which moreover have thestabilities required for routine determinations and reach highconcentration values. For diagnostic purposes, the reliable correlationof pathological process (inflammation, sepsis) with the respectivebiomarker is of primary importance, without there being any need to knowits role in the complex cascade of the endogenous substances involved inthe inflammatory process.

Such an endogenous substance particularly suitable as a sepsis biomarkeris procalcitonin. Procalcitonin is a prohormone whose serumconcentrations reach very high values under the conditions of a systemicinflammation of infectious aetiology (sepsis), whereas it is virtuallyundetectable in healthy persons. High values of procalcitonin are alsoreached in a relatively early stage of a sepsis so that thedetermination of procalcitonin is also suitable for early diagnosis of asepsis and for early distinguishing of a sepsis caused by infection fromsevere inflammations which have other causes. The determination ofprocalcitonin as a sepsis marker is the subject of the publication by M.Assicot et al., “High serum procalcitonin concentrations in patientswith sepsis and infection”, The Lancet, Vol. 341, No. 8844, 1993,515-518; and the patents DE 42 27 454 C2 and EP 0 656 121 B1 and U.S.Pat. No. 5,639,617. Reference is hereby made to said patents and toearly literature references mentioned in said publication forsupplementing the present description. In recent years, the number ofpublications on the subject of procalcitonin has greatly increased.Reference is therefore also made to W. Karzai et al., “Procalcitonin—ANew Indicator of the Systemic Response to Severe Infection”, Infection,Vol. 25, 1997, 329-334; and M. Oczenski et al., “Procalcitonin: a newparameter for the diagnosis of bacterial infection in the peri-operativeperiod”, European Journal of Anaesthesiology 1998, 15, 202-209; andfurthermore H. Redl et al., “Procalcitonin release patterns in a baboonmodel of trauma and sepsis: Relationship to cytokines and neopterin”,Crit Care Med 2000, Vol. 28, No. 11, 3659-3663; and H. Redl et al.,“Non-Human Primate Models of Sepsis”, in: Sepsis 1998;2:243-253; and thefurther literature references cited therein, as typical of recentpublished reviews.

The availability of the sepsis marker procalcitonin has givenconsiderable impetus to sepsis research, and intensive efforts are nowbeing made to find further biomarkers which can supplement theprocalcitonin determination and/or are capable of providing additionalinformation for purposes of fine diagnosis or differential diagnosis.The search for potential new sepsis biomarkers is, however, complicatedby the fact that frequently very little or nothing is known about theexact function or about the exact reasons for the occurrence of certainendogenous substances which are involved in inflammatory or septicprocesses.

The results of the experimental testing of a fruitful purelyhypothetical approach to the determination of further potential sepsismarkers are to be found in DE 198 47 690 Al and WO 00/22439. There, itis shown that, in the case of sepsis, not only is the concentration ofthe prohormone procalcitonin increased but also significantly increasedconcentrations can be observed for other substances which may beincluded among the peptide prohormones. While the phenomenon describedis well documented, the causes of the increase in the concentrations ofprohormones in sepsis are still substantially unexplained.

In the present Application, a result of another fruitful, purelyexperimental approach in the search for further inflammation- orsepsis-specific biomolecules is reported. These experimentalinvestigations, too, originate in the determination of procalcitonin inrelation to systemic inflammatory reactions of infectious aetiology.Thus, it had been observed at a very early stage that the procalcitoninis evidently not formed in the same manner in sepsis as when it is aprecursor for the hormone calcitonin. Thus, high procalcitonin levelswere also observed in patients whose thyroid had been removed. Thethyroid therefore cannot be the organ in which procalcitonin is formedor secreted during sepsis. In the publications by H. Redl et al.,“Procalcitonin release patterns in a baboon model of trauma and sepsis:Relationship to cytokines and neopterin”, Crit Care Med 2000, Vol. 28,No. 11, 3659-3663; and H. Redl et al., “Non-Human Primate Models ofSepsis”, Sepsis 1998; 2:243-253, the results of experimentalinvestigations which are said to be intended for explaining theformation of procalcitonin in sepsis are reported. In said work, anartificial sepsis is produced by endotoxin administration to primates(baboons), and the experimentally produced states in which the highestprocalcitonin concentrations in the blood are reached are determined. Afurther development of the experimental animal model described in saidwork serves, in the context of the present Application, for determiningnovel endogenous sepsis-specific biomarkers of a peptic or proteinnature, the occurrence of which is characteristic for sepsis or certainforms of sepsis and which therefore permit a specific diagnosis ofsepsis. The primate model was chosen because of the very greatsimilarity of the physiology of primates and humans and the highcross-reactivity with many therapeutic and diagnostic human reagents.

Since the endogenous substances formed during inflammations are part ofthe complex reaction cascade of the body, not only are such substancesalso of diagnostic interest but attempts are currently also being made,with considerable effort, to intervene therapeutically in theinflammatory process by influencing the formation and/or theconcentration of individual substances of this type, in order to stop asearly as possible the systemic spread of the inflammation, which spreadis observed, for example, during sepsis. In this context, endogenoussubstances which have been shown to be involved in the inflammatoryprocess are also to be regarded as potential therapeutic targets.Attempts based on certain mediators of the inflammatory process andintended to have a positive therapeutic influence on said process aredescribed, for example, in E. A. Panacek, “Anti-TNF strategies”, Journalfür Anästhesie und Intensivbehandlung; No. 2, 2001, 4-5; T. Calandra etal., “Protection from septic shock by neutralization of macrophagemigration inhibitory factor”, Nature Medicine, Vol. 6, No. 2, 2000,164-170; or K. Garber, “Protein C may be sepsis solution”, NatureBiotechnology, Vol. 18, 2000, 917-918. These therapeutic approaches areintended to lower the concentrations of inflammation-promotingsubstances or to inhibit the formation of such substances, and to do soin particular with the use of specific antibodies (against TNF-α or MIF;cf. E.A. Panacek, “Anti-TNF strategies”, Journal für Anästhesie undIntensivbehandlung; No. 2, 2001, 4-5; T. Calandra et al., “Protectionfrom septic shock by neutralization of macrophage migration inhibitoryfactor”, Nature Medicine, Vol. 6, No. 2, 2000, 164-170) or to increasethe concentration of endogenous substances which have an inhibitoryeffect in the inflammation cascade (Protein C; K. Garber, “Protein C maybe sepsis solution”, Nature Biotechnology, Vol. 18, 2000, 917-918). Thelast-mentioned publication gives an overview of such attempts to have atherapeutic influence on the inflammatory process by influencingselected endogenous target molecules, which attempts have unfortunatelygenerally met with little success to date. In view of the ratherdisappointing therapeutic approaches to date, there is great interest inidentifying further endogenous biomolecules which are as inflammation-or sepsis-specific as possible and which, as therapeutic targets, alsoopen up new prospects for success in fighting inflammation.

The present invention is based on the fact that the enzyme aldose1-epimerase (mutarotase) is detectable in considerable concentrations ininflammations caused by infection in primates and humans, in contrast tohealthy persons, in whom it is not found or is found only inconcentrations at the analytical limit of detection, making saidepimerase suitable both for diagnosis of inflammation/diagnosis ofsepsis and as a novel therapeutic target.

The uses in diagnosis and therapy, which arise because of the occurrenceof aldose 1-epimerase, detected for the first time, in the experimentalsimulation of inflammations or sepsis, are claimed in general form inClaim 1.

Claims 2 to 9 relate to diagnostic uses or methods.

Claim 10 relates in general form to the novel potential therapeuticuses, in particular in the area of the therapy of inflammations andinfections, including sepsis, and Claims 11 to 14 relate to therapeuticapplications of certain pharmaceutical compositions, which applicationshave the aim of influencing the physiological A1E concentrations or A1Eenzyme effect.

As will be explained in more detail below in the experimental section,the invention is based on the fact that, after experimental triggeringof an artificial sepsis in baboons by endotoxin administration (LPS fromSalmonella Typhimurium) and working-up of liver tissue of the treatedanimals by 2D gel electrophoresis, a peptide or protein productidentifiable only in the treated animals was found. This specificproduct was isolated from the electrophoresis gel, trypsin-digested andinvestigated by mass spectrometry. It proved to be aldose 1-epimerase(baboon) on the basis of the identification of two selected peaks whichwere more precisely characterized by tandem mass spectrometry and couldbe assigned to a hypothetical protein to be found in a human cDNAdatabase, and the subsequent identification of this protein bycomparison with the data for a human aldose 1-epimerase fragment or foraldose 1-epimerase from pigs. On the basis of the identity of thesequences identified by mass spectrometry with partial sequences of thesequence data for aldose 1-epimerase which is to be found in databases,the identification of the human equivalent to the isolated protein spotas aldose 1-epimerase is to be regarded as unambiguous according torecognized criteria.

When, in the present Application, the peptide intended for diagnosticpurposes or proposed for therapeutic purposes is referred to as“epimerase”, this does not mean that such an epimerase must be 100%identical to the sequence according to SEQ ID NO:3. Rather, in thepresent Application, epimerase is defined as a peptide which, in thephysiologically occurring form, has partial peptide sequences accordingto SEQ ID NO:1 (FGELPS) and SEQ ID NO:2 (PDGEEGY), and has highhomology, i.e. preferably of more than 60%, more preferably 80%, to thesequence of the human epimerase according to SEQ ID NO:3.

The term epimerase also covers partial peptides of a peptide as definedabove, which partial peptides can be used in particular in thepreparation of reagents, for example selective antibodies, for theepimerase determination or preparation of assays for the epimerasedetermination in biological samples. Those sequences which are obtainedafter deletion of one or more amino acids or short peptide sequencesfrom the peptide according to SEQ ID NO:3 are also to be regarded asepimerase or partial sequences thereof. Furthermore, partial sequences(fragments) suitable for diagnostic and/or therapeutic purposes are inparticular those which comprise a sequence of at least three aminoacids, preferably at least 6 amino acids, of the peptide SEQ ID NO:3.

For particular diagnostic or therapeutic purposes, the epimerasepeptides according to the invention may also be animal peptides, inparticular mammalian peptides, which should have at least 60% homologyto the peptide with SEQ ID NO:3 and, for example, can be used fordiagnostic purposes or in veterinary medicine.

The identification of the protein found only after triggering of sepsisor of inflammation in baboon liver tissue as aldose 1-epimerase(mutarotase; A1E; EC 5.1.3.3.) is of considerable scientific, diagnosticand therapeutic interest, said identification being regarded asunambiguous according to recognized criteria. Epimerase has long beenknown for enzymatic tissue activity. An identification of the human cDNAsequence, determined in the database, as a sequence coding for thecomplete human aldose 1-epimerase has to the best of our knowledge notyet been carried out. The epimerase activity has to date been thesubject of investigations with a primarily scientific objective.Publications by Michael K. Weibel in: Analytical Biochemistry 70,489-494 (1976), or Jane A. Beebe et al., in: Biochemistry 1998, 37,14989-14997 (on galactose mutarotase), and the publications mentionedtherein, may be mentioned here by way of example. In said papers,epimerases are discussed primarily as enzymes which catalyze the mutualconversion of α- and β-anomers of aldoses and which are assumed to beinvolved in sugar transport, e.g. in the kidney and intestine, and insugar metabolism. For medical diagnosis and therapy, aldose 1-epimerasehas played no role to date.

The present invention on the one hand provides protection, according toclaim 1, for the uses stated therein of a peptide which is referred toas epimerase and, according to the above-mentioned definition, isdefined primarily by the presence of the partial sequences of 6 or 7amino acids according to SEQ ID NO:1 and SEQ ID NO:2, respectively,without there being any intention to impose restrictions with regard tothe length and nature of further amino acid sequences. Human A1E isdistinguishable from all other human peptides and proteins which areknown and/or documented in databases by the presence of said two partialsequences. In the complete human form, human aldose 1-epimerase has thesequence according to SEQ ID NO:3.

On the basis of sequence SEQ ID NO:3 now known and the physiologicalrole of human epimerase in combination with the findings on itsincreased formation in inflammations and sepsis, the human epimerase orfragments thereof can be synthesized or prepared by genetic engineeringas recombination products for diagnostic and/or therapeutic purposes bymethods which are now part of the prior art.

Furthermore, the epimerase peptides can be used by known methods of themodern prior art also for producing specific polyclonal and inparticular monoclonal antibodies which are suitable as auxiliaries forthe diagnostic determination of the peptides according to the inventionand/or also as potential therapeutic agents. The production of suitablemonoclonal antibodies against known partial peptide sequences is nowpart of the general prior art.

In the medical diagnostic determination of epimerase according to SEQ IDNO:3 or of selected partial peptides thereof, it is in principlepossible to proceed as described, for example, for the selectiveprocalcitonin determination in P.P. Ghillani et al., “Monoclonalantipeptide antibodies as tools to dissect closely related geneproducts”, The Journal of Immunology, Vol. 141, No. 9, 1988, 3156-3163;and P. P. Ghillani et al., “Identification and Measurement of CalcitoninPrecursors in Serum of Patients with Malignant Diseases”, CancerResearch, Vol. 49, No. 23, 1989, 6845-6851; reference also being madeexpressly and additionally to the immunization techniques describedthere, which represent a possibility for obtaining monoclonal antibodiesalso against partial sequences of epimerase. Variations of thetechniques described and/or further immunization techniques can be foundby a person skilled in the art in relevant standard works andpublications and can be applied in context.

The production of epimerase antibodies using techniques of directgenetic immunization with DNA should furthermore be mentioned expressly.It is furthermore within the scope of the present invention to use, forexample, a cDNA of the desired epimerase or its partial peptides for theimmunization, since it has been found in the past that the spectrum ofthe obtainable antibodies can be extended by such immunizationtechniques. Epimerase according to SEQ ID NO:3 or partial peptidesthereof, for example those which contain the partial sequence SEQ IDNO:l, SEQ ID NO:2 and/or other partial sequences, can, on the basis ofthe available results, serve as specific marker peptides (biomarkers)for the diagnostic detection and for monitoring the course ofinflammations and infections (in particular also of systemic infectionsof the sepsis type). As in the case of the determination ofprocalcitonin, the determination of the epimerase can be effected byusing a method for the differential early diagnosis or for the detectionand for the assessment of the severity and for the therapy-accompanyingassessment of the course of sepsis and infections, in such a method, thecontent of epimerase or of a partial peptide thereof in a sample of abiological fluid or of a tissue of a patient being determined andconclusions being drawn from the established presence and/or amount ofthe peptide determined with regard to the presence of an inflammation,of a severe infection or of a sepsis, and the result obtained beingcorrelated with the severity of the sepsis and the possibilities fortreatment and/or the prospects of treatment being estimated.

Instead of the determination of the epimerase or its fragments oroptionally of posttranslationally modified forms thereof, thedetermination of the associated A1E-mRNA is also possible for diagnosticpurposes. For diagnostic purposes, the epimerase determination can alsobe carried out indirectly as a determination of its enzyme activity inan inflamed organ or tissue or a biological fluid.

It is furthermore possible to carry out the determination of epimeraseas a prognosis marker and marker for the monitoring of the course ofinflammations, in particular systemic inflammations, and sepsis as partof a combination measurement with other markers.

In addition to combination with a procalcitonin measurement, combinationof the measurement of epimerase with the determination of other markersfor sepsis and systemic inflammations is particularly suitable, inparticular with CA 19-9, CA 125, S100B, or S100A proteins involved inthe regulation of inflammations, or with the determination of the novelsepsis markers inflammin (DE 101 19 804.3) and CHP (DE 101 31 922.3)described in the Applicant's prior, unpublished German PatentApplications mentioned below, with the determination of the enzymeglycine N-acyltransferase (GNAT) and/or with the determination ofsoluble cytokeratin fragments, in particular the newly discoveredsoluble cytokeratin-1 fragment (sCYlF; DE 101 30 985.6) and the knowntumour marker CYFRA-21 or TPS and/or one or more of the above-mentionedprohormones. A simultaneous determination of the known inflammationparameter C-reactive protein (CRP) can also be envisaged. On the basisof the novel results described in this and the parallel Applications, acombination with measurements of known biomolecules or biomoleculesstill to be discovered, which are suitable as tissue- or organ-specificinflammation markers, should also be considered generally for finesepsis diagnosis.

The content of said prior Applications of the Applicant is herebyincorporated by reference as part of the disclosure of the presentApplication.

Epimerase or its fragments or fusion products or the DNA coding thereforcan also be used in preventive medicine or therapy. Thus, for example,suitable epimerase fragments can be used for the in vivo production ofepimerase-binding antibodies by active immunization by techniques knownper se. Those molecules which contain the complete epimerase or suitablepartial sequences thereof in posttranslationally modified form, forexample in glycosylated or phosphorylated form, or in a form substitutedby pharmaceutical excipients, e.g. polyethylene glycol radicals, arealso to be regarded as epimerase.

Epimerase or suitable partial sequences thereof may also serve as atarget for therapeutic intervention in the sense that, by means ofsuitable specific binders for epimerase or partial peptides thereof,epimerase is deactivated intracorporeally or is optionally alsoeliminated extracorporeally from the circulation of patients in thesense of a “blood lavage”, or plasmapheresis using suitableimmunoadsorbents or using perfusable solid phases coated with specificbinders for epimerase. As in other known cases in which a pathologicalprocess is influenced by modifying the effect and activity of enzymesinvolved therein, for example the known influencing of infectiousdiseases and colds by medicaments which are cyclooxygenase inhibitors,the action of A1E for therapeutic purposes can also be influenced withthe aid of medicaments which have an enzyme-inhibiting, more preciselyA1E-inhibiting, effect.

In addition to medicaments, in particular specific antibodies,especially humanized monoclonal antibodies, are suitable for the in vivodeactivation of epimerase. Therapeutic influencing of the inflammationcascade can, however, also be effected using the epimerase itself orepimerase agonists or antagonists. Such therapeutic interventions arepossible particularly when further knowledge of the physiologicalfunction of the epimerase in an inflammatory process has been confirmed.It therefore appears that it cannot be ruled out at present thatepimerase plays an important role in the inflammatory process, possiblyby direct or indirect influencing of the pathological process byparticipation in (de)glycosylation steps and other metabolic processesin the activation or deactivation of peptides, proteins, lipids, sugarmolecules and other substances.

The discovery and identification of the epimerase is described in moredetail below, reference being made to the attached sequence listing. Thefigures show the following:

FIG. 1 shows views of 2D electrophoresis gels which permit a comparisonof the spot patterns of cytoplasmic liver cell protein of a healthybaboon (A) with the liver cell proteins of a baboon 5 h after a sepsisinduced by LPS administration (B). The arrow shows the position of thesepsis-specific product according to the invention, epimerase, which issingled out in diagram (B) by a circle;

FIG. 2 shows the mass spectrum of the complete product A1E separated by2D gel electrophoresis, isolated and then trypsin-digested;

FIG. 3 shows the tandem mass spectrum of the A1E fragment 710.3 fromFIG. 2, which has been subjected to an ESI-MS analysis; and

FIG. 4 shows the tandem mass spectrum of the A1E fragment 738.80 fromFIG. 2, which has been subjected to an ESI-MS analysis.

1. INFECTION SIMULATION BY ENDOTOXIN ADMINISTRATION IN AN ANIMAL MODEL(BABOONS)

On the basis of the experiments carried out with baboons for stimulatingprocalcitonin secretion by endotoxin injections (cf. H. Redl et al.,“Procalcitonin release patterns in a baboon model of trauma and sepsis:Relationship to cytokines and neopterin”, Crit Care Med 2000, Vol. 28,No. 11, 3659-3663; H. Redl et al., “Non-Human Primate Models of Sepsis”,in: Sepsis 1998; 2:243-253), baboons (male, about 2 years old, 27 to 29kg in weight) were each intravenously administered 100 μg of LPS(lipopolysaccharide from Salmonella Typhimurium, source: Sigma) per kgbody weight. 5 to 5.5 h after the injection, the animals were sacrificedby intravenous administration of 10 ml of doletal. Within 60 min aftertheir death, all organs/tissues were dissected and were stabilized byfreezing in liquid nitrogen.

In the further processing, samples of individual frozen tissues (1 g)were mixed with 1.5 ml of buffer A (50 mM Tris/HCl, pH 7.1, 100 mM KCl,20% glycerol) while cooling with nitrogen and were powdered in aporcelain mortar (cf. J. Klose, “Fractionated Extraction of Total TissueProteins from Mouse and Human for 2-D electrophoresis”, in: Methods inMolecular Biology, Vol. 112: 2-D Proteome Analysis Protocols, HumanaPress Inc., Totowa, N.J.). After subsequently centrifuging for 1 hour at100,000 g and +4° C., the supernatant obtained was recovered and wasstored at −80° C. until required for further processing.

Because experiments with the samples obtained as above showed that thelargest amount of procalcitonin is found in liver tissue of treatedanimals, protein extracts from baboon liver were employed in the searchfor novel sepsis-specific biomarkers.

2. Proteome Analysis Using Cytoplasmic Liver Cell Proteins of Baboons

Cytoplasmic liver cell protein extracts of healthy baboons on the onehand (control) and, on the other hand, baboons which had been injectedwith LPS were used in a proteome analysis. In the initial analytical 2Dgel electrophoresis, liver extract, containing 100 μg of protein, wasstandardized to 9 M urea, 70 mM DTT, 2% ampholyte pH 2-4 and thenseparated by means of analytical 2D gel electrophoresis, as described inJ. Klose et al., “Two-dimensional electrophoresis of proteins: Anupdated protocol and implications for a functional analysis of thegenome”, Electrophoresis 1995, 16, 1034-1059. The visualization of theproteins in the 2D electrophoresis gel was effected by means of silverstaining (cf. J. Heukeshoven et al., “Improved silver staining procedurefor fast staining in Phast-System Development Unit. I. Staining ofsodium dodecyl gels”, Electrophoresis 1988, 9, 28-32).

For the evaluation, the protein spot patterns of the samples fromuntreated animals were compared with the protein spot patterns whichresulted from liver tissue samples of treated animals. Substances whichoccurred in no control sample but additionally occurred in all treatedanimals were selected for further analytical investigations. FIG. 1shows a comparison of the 2D electrophoresis gels for a control sample(A) and a sample from a treated animal (B), the additional protein spotin (B) corresponding to epimerase, the position of which is singled outby an arrow and a circle.

The novel specific proteins identified in the protein spot pattern ofthe analytical 2D gel electrophoresis were subsequently prepared bymeans of preparative 2D gel electrophoresis using 350 ug of protein (cf.once again (10)). In the preparative 2D gel electrophoresis, thestaining was effected by means of Coomassie Brilliant Blue G250 (cf. V.Neuhoff et al., “Improved staining of proteins in polyacrylamide gelsincluding isoelectric focusing gels with clear background at nanogramsensitivity using Coomassie Brilliant Blue G-250 and R-250”,Electrophoresis 1988, 9, 255-262).

The protein spot preselected for the further analysis was cut out of thegel. It was trypsin-digested using the method which is described in A.Otto et al., “Identification of human myocardial proteins separated bytwo-dimensional electrophoresis using an effective sample preparationfor mass spectrometry”, Electrophoresis 1996, 17, 1643-1650, and thenanalyzed by mass spectroscopy, in particular using mass spectrometricinvestigations as described and discussed, for example, in G. Neubaueret al., “Mass spectrometry and EST-database searching allowscharacterization of the multi-protein spliceosome complex”, in: naturegenetics vol. 20, 1998, 46-50; J. Lingner et al., “Reverse TranscriptaseMotifs in the Catalytic Subunit of Telomerase”, in: Science, Vol. 276,1997, 561-567; M. Mann et al., “Use of mass spectrometry-derived data toannotate nucleotide and protein sequence databases”, in: TRENDS inBiochemical Sciences, Vol. 26, 1, 2001, 54-61. The trypsin-digestedsamples were subjected to tandem mass spectrometry after an ESI(ElectroSpray Ionization). A Q-TOF mass spectrometer having a so-callednanoflow-Z-spray ion source from Micromass, UK, was used. The workinginstructions of the apparatus manufacturer were followed.

3. Identification of Epimerase

As shown in FIGS. 1(A) and 1(B), liver cell extracts of baboons to whichan LPS injection had been administered contain, inter alia, a novelprotein for which a molecular weight of about 40000±2000 Dalton wasestimated on the basis of the gel electrophoresis data in comparisonwith marker substances having a known molecular weight, while anisoelectric point of about 6 to 6.4 was estimated from the relativeposition of the protein from the first dimension.

This protein was analyzed by mass spectrometry, FIG. 2 showing the massspectrum of the total trypsin-digested protein.

Fragments of the “parent spectrum” according to FIG. 2 were identifiedby tandem mass spectroscopy. The mass spectra obtained for two of thesefragments are shown in FIG. 3 and 4. The fragments could be identifiedcomputationally in a manner known per se as the partial peptidesequences SEQ ID NO:1 and SEQ ID NO:2.

The partial sequences according to SEQ ID NO:1 and SEQ ID NO:2identified by tandem mass spectrometry were then compared with theprotein sequences which were available in sequence databases. An aminoacid sequence for a hypothetical human protein comprising 342 aminoacids with the theoretical molecular weight of 37765 Dalton (cf. SEQ IDNO:1), in which the partial sequences according to SEQ ID NO:1 and SEQID NO:2 were contained in the positions 9 to 14 and 134 to 140,respectively, was found in the cDNA database NCBI, Accession No.AAH14916. By sequence comparison with two amino acid sequences which areto be found using NiceProt View of TrEMBL and of which one is of humanorigin and comprises 204 amino acids (Entry name/Primary accession No.Q12915) and the other originates from pig kidneys (Entry name/Primaryaccession No. Q9GKX6), and both of which are assigned to aldose1-epimerase, it was possible to identify the sequence according to SEQID NO:3 as an amino acid sequence of human aldose 1-epimerase.

The complete sequence of the human epimerase which is thus knownexplicitly makes it possible to prepare human epimerase or any partialsequences (fragments) thereof in a targeted manner for purposes of humanmedicine (diagnosis or therapy), and to do so using known synthetic orgenetic engineering methods for the preparation of peptides. The sameapplies for purposes of veterinary medicine, it being possible in thesecases to rely on corresponding known, e.g. bovine, epimerase sequencesor it being possible easily to discover animal-specific epimerasesequences in corresponding animal-specific databases on the basis of theanalogies with the known bovine and human sequences. Such peptides canthen serve, for example in analogy to the procedure described in P.P.Ghillani et al., “Monoclonal antipeptide antibodies as tools to dissectclosely related gene products”, The Journal of Immunology, Vol. 141, No.9, 1988, 3156-3163; and P. P. Ghillani et al., “Identification andMeasurement of Calcitonin Precursors in Serum of Patients with MalignantDiseases”, Cancer Research, Vol. 49, No. 23, 1989, 6845-6851, forproviding suitable antibodies, in particular monoclonal antibodies,which in turn permit the provision of assays for the immunodiagnosticdetermination of epimerase or selected partial peptides thereof.

Monoclonal antibodies obtainable in the known manner described can alsoserve, particularly after humanization known per se, for the developmentof novel therapeutic agents (cf. the therapeutic approaches summarizedin K. Garber, “Protein C may be sepsis solution”, Nature Biotechnology,Vol. 18, 2000, 917-918). Furthermore, an in vivo neutralization of theepimerase is also possible by blocking the expression of the epimerasegene. The therapeutic interventions arising from the discoveries in thepresent Application also include administration of active substanceswhich inhibit the enzyme activity of the epimerase.

It is furthermore within the scope of the present invention to useepimerase itself or partial peptides thereof as pharmaceutical activesubstances. The invention consequently also relates to pharmaceuticalcompositions which contain, as the actual active substance, one of thepeptides according to the invention or antibodies produced against thesepeptides and prepared for administration to patients, together with asuitable pharmaceutical carrier.

1. Use of aldose 1-epimerase (A1E) from body fluids or body tissues inhuman and veterinary medicine as a marker peptide for diagnosis, forprognosis of the course and for monitoring of the course ofinflammations and infections and/or as a target for therapeuticallyinfluencing the course of inflammations and/or infections.
 2. Use ofaldose 1-epimerase according to claim 1 in differential early diagnosisand detection, for prognosis of the course, for assessment of theseverity and for therapy-accompanying assessment of the course of sepsisand severe infections, in particular sepsis-like systemic infections, bydetermination of the occurrence and/or of the amount of aldose 1-epimerase in a biological fluid or a tissue sample of a patient. 3.Method for differential early diagnosis and detection, for prognosis ofthe course and assessment of the severity and for therapy-accompanyingassessment of the course of sepsis and severe infections, in particularsepsis-like systemic infections, characterized in that the presenceand/or amount of human aldose 1 -epimerase (SEQ ID NO:3) in a biologicalfluid or a tissue sample of a patient is determined and conclusions aredrawn from the detectability and/or amount thereof with regard to thepresence, the expected course, the severity or the success of a therapyof the sepsis or of infection.
 4. Method according to claim 3,characterized in that it is an immunodiagnostic assay method.
 5. Methodaccording to claim 3, characterized in that the determination of aldose1-epimerase is carried out indirectly as a determination of theassociated A1E-mRNA or of the A1E enzyme activity.
 6. Method accordingto claim 3, characterized in that it is carried out as part of amultiparameter determination in which at least one further sepsisparameter is simultaneously determined and a measured result in the formof a set of at least two measured quantities, which is evaluated forfine sepsis diagnosis, is obtained.
 7. Method according to claim 6,characterized in that, in the course of the multiparameterdetermination, at least one further parameter which is selected from thegroup consisting of procalcitonin, CA 19-9, CA 125, S100B, S100Aproteins, soluble cytokeratin fragments, in particular CYFRA 21, TPSand/or soluble cytokeratin 1 fragments (sCY1F), the peptides inflamminand CHP, peptide prohormones, glycine N-acyltransferase (GNAT) and theC-reactive protein (CRP) is determined in addition to aldose 1-epimerase.
 8. Method according to claim 6, characterized in that themultiparameter determination is carried out as a simultaneousdetermination by means of a chip technology measuring apparatus or animmunochromatographic measuring apparatus.
 9. Method according to claim8, characterized in that the evaluation of the complex measured resultobtained by means of the measuring apparatus is carried out with the aidof a computer program.
 10. Use of aldose 1 -epimerase according to claim1 as a means for prophylactic or therapeutic influencing of the courseof inflammatory diseases and infections, including sepsis. 11.Pharmaceutical composition for the treatment of inflammations, includingsystemic inflammations, characterized in that it comprises aldose1-epimerase or a functional partial peptide thereof and apharmaceutically acceptable carrier.
 12. Pharmaceutical composition forthe treatment of inflammatory and other stress reactions of the body,characterized in that it contains antibodies produced against aldose1-epimerase or aldose 1-epimerase fragments or processed aldose 1-epimerase and prepared for administration to patients, together with asuitable pharmaceutical carrier.
 13. Pharmaceutical composition for thetreatment of inflammatory diseases of the body, in particular systemicinflammations, or of inflammations caused by infection, in which thereis the danger of a systemic spread, characterized in that it contains anactive substance which completely or partly inhibits the enzyme activityof aldose 1-epimerase and a suitable pharmaceutical carrier. 14.lmmunoadsorbent which has a solid phase for the selective binding ofaldose 1-epimerase from the blood circulation of a patient, for thetreatment of inflammations and sepsis.
 15. Method according to claim 7,characterized in that the multiparameter determination is carried out asa simultaneous determination by means of a chip technology measuringapparatus or an immunochromatographic measuring apparatus.